The Targeted Proteomics Core designs and implements targeted assays that have been used increasingly by Center investigators to quantitate and validate potential protein biomarkers initially identified from the protein profiling analyses carried out by the Discovery Proteomics Core (DPC), genomic studies, including the use of neuronal cell type-specific transcriptional and translational profiling, or the literature. These analyses will include relative and absolute quantitation of targeted protein levels or alterations in their post-translational modifications that have been implicated in model and other organisms as adaptive changes that occur in response to drugs of abuse. To address the huge level of cellular and sub-cellular heterogeneity in the central nervous system, another objective is to develop the targeted workflows needed to analyze subcellular organelles and sub-proteomes from the single types of neurons that define the circuits that underlie the actions and addictive properties of drugs of abuse. In Aim 1 we will integrate high resolution Data-Independent Acquisition (DIA) into our large scale targeted proteome assays using fractionated Data-Dependent Acquisition (DDA) peptide libraries generated by the DPC from mouse, rat, and human brain regions. DIA analysis has the potential to change the proteomic landscape since each sample only needs to be run once, and then all peptide fragments can retrospectively be identified and quantified. In Aim 2 we will develop targeted, highly sensitive DIA assays to examine the proteomes of specific neuronal sub-types and their organelles (e.g., nuclei, synaptic vesicles), sub-cellular fractions or partially enriched samples (e.g., PSD) and then use Parallel Reaction Monitoring (PRM) to validate the most significantly differentially expressed proteins. In support of this Aim, we will develop improved methodologies for protein profiling of the limiting amounts of protein that can be obtained from the LCM and FACS-based approaches needed to isolate neuronal sub-types. Since eukaryotic proteins almost always are assembled into multiprotein complexes in vivo, Aim 3 will leverage the data from our targeted assays by implementing improved proximity biotinylation technologies for identifying protein interactomes for those proteins that are found to be most differentially expressed by our targeted assays. To further enhance the interpretation of the data from our targeted proteome analyses, in Aim 4 we will implement a Puromycin-associated Nascent CHain Proteomics (PUNCH-P) assay for translatome profiling. Since under steady state conditions protein levels are largely determined by transcript concentrations, it will be of considerable interest to compare the approximately steady state protein levels determined by our DIA analyses with the nascent protein levels determined by our PUNCH-P analyses before and after exposure to drugs of abuse. In Aim 5, we will continue to train Neuroproteomics Center members in targeted mass spectrometric techniques including experimental design, sample preparation and handling, digestion protocols, interpretation of PRM and DIA spectra so they can optimally utilize the advanced technologies available in the Center.